Method of determining skin tissue cholesterol

ABSTRACT

A method of determining skin tissue cholesterol includes preparing a section of skin surface, applying a dosed amount of an enzyme-containing mixture onto the skin, exposing, evaluating the concentration of cholesterol in a reaction solution and calculating the concentration of skin cholesterol, wherein the area of contact of the skin with the mixture is limited on the section of the skin surface by means of a sealing vessel without a bottom with a base fixed on the skin, a mixture comprising 2.0-2.5 units of cholesteroloxidase, 0.04-0.06% by weight of sodium dezoxycholate, 0.1-0.2.% by weight of 3-(dodecyl-dimethyl-ammonium)-propanesulfonate in a buffer solution with pH=6.8 is used as the mixture, evaluating the concentration of cholesterol in the reaction solution is carried out by measuring the level of hydrogen peroxide. Measurements of the level of hydrogen peroxide may be carried out by immersing an electrochemical sensor or a colorimetric indicator in the reaction solution.

FIELD OF THE INVENTION

The invention relates to the field of medicine, more exactly to a methodof examining the skin with biochemical methods. The invention may alsobe used in cosmetology, pharmacology, criminalistics, etc., to determinethe content of cholesterol, both free and total, in the skin.

BACKGROUND ART

In clinical conditions the determination of cholesterol in the skinmakes it possible in a noninvasive manner, i.e. without taking bloodsamples or biopsy material, to evaluate the level of tissue cholesterol.

A method is known for determining the presence of different chemicalsubstances on the surface of the skin, these substances being in theblood channel of a human or animal (glucose, alcohol, lactate,hypoxanthine). The method includes the subcutaneous administration of anenzyme immobilized on a silicon surface, heating a section of the skinto 38-44° C. and registering a local change in the concentration ofoxygen or hydrogen peroxide on the skin by means of an electrochemicalsensor (U.S. Pat. No. 4,458,686, A 61 B 5/00, published July 1984).

A drawback of the known method is the complexity of subcutaneousadministration of an immobilized enzyme, the formation of hydrogenperoxide directly in tissues, which may result in undesirableconsequences (the transition of hemoglobin into methemoglobin)—thedevelopment of hypoxia.

A method is known for determining skin cholesterol by taking samples oftissue with subsequent extraction of cholesterol. The method makes itpossible to determine not only the amount of total cholesterol, butseparately free and esterified (H. Bouissou, M. Th. Pieraggi, M. Julian,Identifying arteriosclerosis and aortic ateromathosis by skin biopsy.Atherosclerosis, v. 19, pp. 449-458).

A development of this method is an extraction method, in accordance withwhich extraction of cholesterol is carried out without taking a tissuesample directly on a section of live skin. A mixture of ethyl alcoholand ether is used as the extractant. A test tube with the extractant isheld against the surface of the skin for several minutes. The lipidswhich are in the surface layers of the skin dissolve and pass into theextractant solution, after which evaporation of the solvent is carriedout and the substances passing into a liquid phase are determined (Yu.M. Lopukhin. The skin and atherosclerosis [a three-drop test]. 1992,Gordon and Breach Science Publishers S.A. UK).

The content of cholesterol in the epidermis reflects the accumulation ofcholesterol in the skin and correlates with the content of the latter inthe aorta, and also with the area of damage of the aorta in the case ofatherosclerosis (Yu. M. Lopykhin. The skin and atherosclerosis [athree-drop test], 1992, Gordon and Breach Science Publishers S.A. UK).Thus, determination of the content of cholesterol in the skin makes itpossible to obtain unique information on the state of the tissue pool ofcholesterol, and this is extremely important from both the point of viewof early diagnosis of atherosclerosis (preclinical state) and for thepurpose of monitoring the treatment of patients with atherosclerosis.

In pharmacology, the method of determining cholesterol in the skin makesit possible to carry out evaluation of the effectiveness of drugs whichaffect both the synthesis of cholesterol in tissues (a group ofstatins), and its removal from the organism by means of differentsorbents.

A drawback of the known method is the necessity of carrying out abiopsy, contact of skin with the solution of extractants, and also thelengthy time necessary to carry out the analysis.

The method most similar to the proposed method is the method ofdetermining skin cholesterol including preparing a section of skinsurface, applying onto the skin (or another cholesterol-containingsurface) a dosed amount of a solution that is an affino-enzymaticconjugate of a general structure A—C—B, wherein A is a compound capableof linking with cholesterol (affinate), C is a linking bridge orpolymer, B is an enzyme providing a color product as a result ofconversion of an according substrate. The amount of cholesterol isdetermined according to the intensity of the transition of the dye to adyed form (U.S. Pat. No. 5,489,510, A 61 B 10/00, published February1996—prototype).

Drawbacks of this method are the limited field of use, i.e. theimpossibility of determining esterified cholesterol, the low specificityof the analysis, in particular interaction of the conjugate not onlywith cholesterol, but also with other lipids.

DISCLOSURE OF THE INVENTION

The object to the attainment of which the instant invention is directedis to increase the specificity, simplify and expand the field ofutilization of the method, increase the accuracy of determination ofcholesterol in the skin of a human and other condensed mediums.

The stated object is attained in a method of determining skincholesterol, including preparing a section of a surface, applying ontothe skin surface a dosed amount of an aqueous solution of an enzyme withaddition of a surfactant, exposing, evaluating the concentration ofcholesterol in a reaction solution and calculating the concentration ofskin cholesterol, in that in accordance with the proposal, afterpreparing the section of the surface, an area of contact of the skinwith the enzyme solution and the surfactant is limited by means of anappliance, the enzyme solution with the surfactant for determination offree tissue cholesterol has the following composition (% by weight):

cholesteroloxidase 2.0-2.5 units sodium dezoxycholate 0.04-0.063-(dodecyl-dimethyl-ammonium)- 0.1-0.2 -propanesulfonate phosphatebuffer pH 6.8 balance to 100%,

while in order to determine the total cholesterol, the composition hasthe following makeup (% by weight);

cholesteroloxidase 2.0-2.5 units cholesterolesterase 3-5 units sodiumdezoxycholate 0.04-0.06 3-(dodecyl-dimethyl-ammonium)- 0.1-0.2-propanesulfonate phosphate buffer pH 6.8 balance to 100%,

exposing the solution on the skin is carried out for 2 minutes todetermine free cholesterol and for 10 minutes to determine totalcholesterol, while evaluation of the concentration of cholesterol iscarried out by direct measurement of the concentration of hydrogenperoxide in the reaction solution. Limitation of the area of contact ofthe skin with the surfactant solution is carried out using a sealedcuvette without a bottom with a base fixed on the skin.

Cholesteroloxidase, obtained from different sources (Brevibacterium sp.,Nocardia erythropolis), is used. The hydrogen peroxide generated duringinteraction of cholesterol with cholesteroloxidase is determined bymeans of an electrochemical sensor or by using spectrophotometricequipment.

The cholesteroloxidase catalyzes oxidation of cholesterol with theparticipation of molecular oxygen and the formation of hydrogenperoxide:

cholesterol+O₂=4-cholestine-3-OH+H₂,O₂

When a mixture of a surfactant—ionic detergent (sodium dezoxycholate[DOC])—in a concentration of 0.04-0.06% and a zwitterion detergent -3-(dodecyl-dimethyl-ammonium)-propanesulfonate (DAPS) in a concentrationof 0.1-0.2% is used, optimum speed and completeness of oxidation ofcholesterol are detected. This reaction system makes it possible toenhance the effectiveness of action of cholesteroloxidase as a result ofreduction of the inhibiting action of the surfactant and to reduce theconsumption of expensive detergents. Such a composition of detergentsensures access to free cholesterol and a high speed of its oxidation.This makes it possible to use relatively low concentrations ofcholesteroloxidase in the determination of free cholesterol. When akinematic variant of measurement is used, only 4-8 nmols per ml ofreaction mixture, and during determination according to the final pointto carry out complete oxidation of cholesterol in 10 minutes, 20 nmolsof enzyme per ml of reaction mixture are sufficient.

Photometric methods make it possible to achieve the lower limit ofdetecting oxidase substrates of about 10⁻⁶M.

An electrochemical sensor with cholesteroloxidase, immobilized on acollagen membrane, makes it possible to determine the concentration ofcholesterol from 10⁻⁴ to 0.08 mM (USSR patent 622424, G 01 N 33/16,1976. Bertand et al. 1981. Multipurpose electrode with different enzymebound to collagen films. Analyt. Chim. Acta. V.126, pp. 23-34).

TABLE 1 Sodium dezoxycholate (DOC), % DAPS, 0 0.02 0.04 0.06 0.08 % V ODV OD V OD V OD V OD 0    0 0     0 0     0 0     0 0     3 0.08  0.10 270.067 51 0.08  47 0.071 41 0.069 33 0.068 0.15 cloudy solution 58 0.10853 0.118 41 0.109 38 0.093 0.20 cloudy 72 0.103 63 0.112 51 0.108 450.103 0.25 cloudy 74 0.110 65 0.105 53 0.108 44 0.097

Data are presented in Table 1 which were obtained during oxidation offree cholesterol contained in a control solution of cholesterol withdifferent ratios of the two indicated detergents. The results arepresented in units of optical density (OD) and the speed of reaction ofoxidation according to the value of the angle of inclination of thekinetic curve (V).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a sealed cuvette without a bottom.

FIG. 2 shows the application of a dosed amount of solution.

FIG. 3 shows an electrochemical electrode brought into contact with thesolution.

VARIANTS OF CARRYING OUT THE INVENTION

The method is realized in the following manner. At first a section ofthe skin on which measurement is to be carried out is rubbed with atampon moistened with ethyl alcohol. A special appliance is fixed on theprepared section of skin surface—a sealed cuvette without a bottom (FIG.1), where 1 is a cuvette with a thread for fixing an electrochemicalsensor or lid; 2 is a substrate, ensuring fixation of the cuvette andits air-tightness on the skin surface. The cuvette makes it possible tolimit the area of skin surface (1 cm²), where the analysis is to bemade, and to apply onto that section (FIG. 2) a dosed amount (volume 0.1 ml) of a solution of a mixture of an enzyme with a surfactant.

Exposition of the solution on the skin is carried out within twominutes. During the exposition of the solution comprising the mixture ofenzyme and the surfactant, hydrogen peroxide is generated, theconcentration of which is directly proportional to the content ofcholesterol. After exposition the solution containing hydrogen peroxideis carried to an electrochemical electrode where measurement of theconcentration of the generated hydrogen peroxide takes place.

A variant of the method is proposed in which an electrochemicalelectrode is brought into contact with the solution in the cuvettedirectly on the surface of the skin (FIG. 3).

In the case where a spectrophotometric method of measuring theconcentration of hydrogen peroxide is used, a solution comprisinghydrogen peroxide is carried into the cuvette of a photometer where thedeveloping system (peroxidase enzyme and substrate). The concentrationof the peroxide is determined by the value of the optical density of thesolution, which is directly proportional to the concentration ofhydrogen peroxide. Determination of the concentration of hydrogenperoxide in a liquid phase with the use of a photometer is preferable inlaboratory conditions. Outside the laboratory, for example, in homediagnosis, it is possible to use methods with the use of “dry” chemistrytechnology. In order to do this colorimetric indicators on paper stripsmay be used, which make it possible to determine the concentration ofhydrogen peroxide in the range of from 0.2 to 20 mg/l that fully coversthe necessary range of concentrations.

When working with a colorimetric indicator (strip), the paper strip isimmersed in a solution of exposed enzyme and surfactant, which is on theskin surface in a cuvette. After immersion, the change in the color ofthe strip is evaluated, which is also determined by the concentration ofthe generated hydrogen peroxide or by means of a color scale or by meansof a portable reflective photometer.

Determination of the amount of cholesterol on the skin surface is notonly possible according to the amount of hydrogen peroxide generated inthe course of the oxidation reaction of cholesterol with ancholesteroloxidase enzyme, but also according to the value ofconsumption of oxygen in the course of the reaction, and also the valueof oxidized cholesterol—cholestenone. The technology of determininghydrogen peroxide is preferable from the point of view of convenienceand relative simplicity.

Not only the determination of free, but also of esterified cholesterolis important in diagnosis, since their ratio changes in a number ofdiseases. Determination of esterified cholesterol makes it possible tocarry out the determination of total cholesterol, which is the sum offree and esterified cholesterol. With this object in mind, we carriedout enzymic hydrolysis of cholesterol esters with cholesterolesteraseenzyme obtained both from tissue of the pancreatic gland and frommicrobic origin. In order to determine esterified cholesterol on skinsurface, a solution of a mixture of enzymes—cholesterolesterase withcholesteroloxidase, and a surfactant was applied on the skin surface.The solution is exposed for 10 minutes after which determination ofhydrogen peroxide is carried out with one of the methods indicatedabove.

EXAMPLE 1

Determination of free cholesterol in healthy volunteers by means of anelectrochemical sensor. A cuvette shown in FIG. 1 is placed on the skinsurface of a palm (tenar region). The cuvette makes it possible to fix asolution of an enzyme and a surfactant with a volume of 0.1 ml on a 1 cmskin surface. At first the skin surface is rubbed with a solution ofethyl alcohol. After the cuvette is set in place, a solution of thefollowing composition (% by weight) is poured therein:

cholesteroloxidase 2.0-2.5 units sodium dezoxycholate 0.063-(dodecyl-dimethyl-ammonium)- 0.2 -propanesulfonate phosphate buffer pH6.8 balance to 100%.

After incubation of the solution oil the skin surface for two minutes,0.05 ml of the solution are taken off and carried to the measuringsurface of an electrochemical sensor. The result—a current valueproportional to the content of the generated hydrogen peroxide, is readafter 5 seconds. A recalculation device makes it possible to show on aliquid crystal screen the value of the content of free cholesterol onskin surface expressed either in μmol/1 or in μg/cm².

Determination of cholesterol using method (4) makes it possible todetect one, two or three dyed spots. The results of simultaneousconduction of these two tests are presented in Table 2. It is evidentthat deviations in the content of free cholesterol in the skin with achange by almost two times from 10.1 to 5.5 μmol/1 provide a positivereaction of one cross, while at a cholesterol concentration of 9.3μmol/1 the fourth test showed a reaction of two positive crosses (++).

TABLE 2 Free cholesterol, Three-drop test in No. of test μmol/l crosses1 10.1  (+) 2 6.8 (+) 3 5.5 (+) 4 9.3 (++) 5 7.1 (+)

EXAMPLE 2

Determination of free cholesterol in patients with stenosingatherosclerosis with clinical manifestations of ischemic heart disease(IHD). When the technology described in Example 1 is used, the level offree cholesterol in the skin of patients with IHD varies within therange of from 18.7 to 35.8 μmol/1 (see Table 3).

TABLE 3 No. of IHD patient Free cholesterol, μmol/l Three-drop test incrosses 1 22.5 (++) 2 18.7 (++) 3 27.4 (+++) 4 28.7 (+++) 5 31.9 (+++) 620.7 (+++) 7 26.3 (++) 8 25.8 (++) 9 35.8 (+++) 10  20.1 (++) 11  25.0(++) 12  24.9 (+++) 13  29.1 (++) 14  21.1 (++)

EXAMPLE 3

Determination of free cholesterol by means of a special paper strip. Acuvette is placed on the skin surface of the palm (tenar region), makingit possible to fix a volume of a solution of an enzyme and a surfactantequal to 0.1 ml on a 1 cm² skin surface. The skin surface is firstrubbed with a solution of ethyl alcohol. After the cuvette has been putin place a solution of cholesteroloxidase and a surfactant is pouredtherein. After incubation for two minutes, a special paper strip ismoistened therein. The result of determination of the content of freecholesterol of the skin is calculated by means of a calibration colorscale, or according to the reading of a reflective photometer.

EXAMPLE 4

Determination of total cholesterol (the sum of free and esterified) ofskin cholesterol. A cuvette is placed on the skin surface of the palm,fixing a predetermined volume of the following solution on the skinsurface:

cholesteroloxidase 2.0-2.5 units cholesterolesterase 3-5 units sodiumdezoxycholate 0.04 3-(dodecyl-dimethyl-ammonium)- 0.1 -propanesulfonatephosphate buffer pH = 6.8 balance to 100%.

After that the cuvette is closed with a special lid, since the time ofexposition in that case is 10 minutes and evaporation may take place inthat time, which would result in concentration of the solution. On theaverage, in the control group, the content of total cholesterol exceedsby 10-20% the value of free cholesterol, i.e. the value of esterifiedcholesterol is 10-20% of the total skin cholesterol.

What is claimed is:
 1. A method of determining cholesterol present onskin surface, comprising the steps of: a. providing an open-ended vesselhaving a base; b. providing an enzyme solution comprising 2.0 to 2.5units of cholesterol oxidase, 0.04 to 0.06 weight % of sodiumdezoxycholate, and 0.1 to 0.2 weight % of3-dodecyl-dimethyl-ammonium-propanesulfonate, buffered to a pH of 6.8 inwater; c. bringing said vessel into contac with the skin surface in amanner such that the base of said vessel sealingly engages the skinsurface, thereby delimiting an area of said skin surface within saidvessel; d. adding a predetermined amount of said enzyme solution intosaid vessel so that said enzyme solution contacts the skin surface overthe delimited area thereof; e. allowing said enzyme solution to remainin contact with the skin surface over the delimited area thereof for aperiod of time sufficient for the cholesterol oxidase to catalyzeoxidation of cholesterol and cause formation of hydrogen peroxide; and,f. measuring the amount of hydrogen peroxide formed in step (e), theamount of hydrogen peroxide measured being indicative of theconcentration of skin cholesterol.
 2. A method according to claim 1wherein said enzyme solution further comprises 3 to 5 units ofcholesterol esterase.
 3. A method according to claim 2, wherein said 3to 5 units of cholesterol esterase are added to said enzyme solution insaid vessel between steps (d) and (e).
 4. A method according to claim 1,wherein step (f) is carried out by spectrophotometry after the additionof peroxidase and a substrate to said enzyme solution in said vessel. 5.A method according to claim 1, wherein step (f) is carried out by meansof an electrochemical sensor that is immersed into said enzyme solution.6. A method according to claim 1, wherein step (f) is carried out bymeans of a colorimetric indicator immersed into said enzyme solution.